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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 13, 2015 |
| Title |
HuR KO_Ex vivo2 |
| Sample type |
SRA |
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| Source name |
Splenic B cells
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| Organism |
Mus musculus |
| Characteristics |
strain: HuRflox/flox x mb1cre
genotype: HuR KO
cell type: Splenic B cells negative selection using a cocktail of biotin-conjugated antibodies against CD43, CD4 , and Ter-119 (Mylteni 130-090-862)
condition: Ex-vivo
illumina barcode: AGTCAA
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| Treatment protocol |
After isolation, splenic B cells were cultured in RPMI 1640 Medium (Dutch Modification) plus 5% FCS, antibiotics, 2 mM L-glutamine and Beta-mercaptoethanol (5 microMolar) at a density of 5x10^5 cells/ml. They were activated with LPS (10 micrograms/ml) for 48 hours.
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| Growth protocol |
HuRflox/flox x mb1wt and HuRflox/flox x mb1cre mice were littermates. This mouse line was maintained under Babraham Institute AWEEC and UK home Office regulation.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA extraction using TriZol (life Technologies)
Illumina TruSeq mRNA sample prep kit (Barcodes used for sample multiplexing: ATGTCA, CAGATC, CGATGT, GTGAAA, TGACCA, CCGTCC, ACAGTG, GCCAAT, CTTGTA, AGTCAA)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1000 |
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| Description |
supplementary file: RNAseq Analysis_DESeq_Ex vivo_B_cells.txt
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| Data processing |
Raw sequence reads were trimmed to remove both barcodes and adapters using Trim Galore (www.bioinformatics.babraham.ac.uk/projects/trim_galore/) (v0.3.3, default parameters).
Remaining sequences were mapped to the mouse GRCm38 genome using Tophat (Bioinformatics. 2009 May 1;25(9):1105-11. and http://ccb.jhu.edu/software/tophat/index.shtml). Read counting was performed using HTSeq. Ensembl annotation GRCm38.72 was used for gene or exon annotation. Intron definition from Ensembl exon annotation was performed using custom scripts.
Differential gene expression analysis was performed using DESeq (Anders, S., and Huber, W. (2010). Differential expression analysis for sequence count data. Genome biology 11, R106). Ensembl annotation GRCm38.72 was used for differential analysis.
Differential exon usage analysis was performed using DEXSeq (Anders, S., Reyes, A., and Huber, W. (2012). Detecting differential usage of exons from RNA-seq data. Genome research 22, 2008-2017). Libraries generated from LPS-activated B cells were used. Ensembl annotation GRCm38.72 was used for differential analysis.
Differential intron usage analysis was performed using DEXSeq (Anders, S., Reyes, A., and Huber, W. (2012). Detecting differential usage of exons from RNA-seq data. Genome research 22, 2008-2017). Libraries generated from LPS-activated B cells were used. Ensembl annotation GRCm38.72 was used for differential analysis.
Genome_build: GRCm38
Supplementary_files_format_and_content: Files contain the report generated by DESeq and DEXSeq after differential expression analysis.
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| Submission date |
Oct 07, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Felix Krueger |
| E-mail(s) |
fkrueger@altoslabs.com
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| Organization name |
Altos Labs
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| Department |
Bioinformatics
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| Street address |
Granta Park
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| City |
Cambridge |
| ZIP/Postal code |
CB21 6GP |
| Country |
United Kingdom |
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| Platform ID |
GPL15103 |
| Series (2) |
| GSE62129 |
HuR- dependent regulation of mRNA splicing is essential for the B cell antibody response [RNA-Seq] |
| GSE62148 |
HuR- dependent regulation of mRNA splicing is essential for the B cell antibody response |
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| Relations |
| Reanalyzed by |
GSE80797 |
| BioSample |
SAMN03098039 |
| SRA |
SRX726825 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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